The present invention relates to a method and kit for the diagnosis of Schistosomiasis in human sample matter by Polymerase Chain Reaction (PCR). The method is based on the detection of the parasite DNA and is especially useful in cases of low infection intensity, for which parasitological stool tests (Kato-Katz) demonstrate little sensitivity.
Schistosomiasis is a disease caused through infection by parasites of genus Schistosoma, with Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobiom being the principal infectant species. The disease has been recorded for 4000 years and still constitutes an important problem for public health, affecting more than 200 million people in undeveloped countries. The species S. mansoni is endemic in 54 countries and territories of South America, the Caribbean, Africa and the eastern Mediterranean region.
These helminths, digenetic trematodes, infect man by penetration of the skin by the cercariae (larval stage), that are eliminated in the water by different species of molluscs, their intermediate hosts. After migrating through the skin, the larvae of S. mansoni traverse the lungs and attain the hepatic portal system, then subsisting in the veins and plexus of the gastrointestinal tract, where they become adult and mate, following which the female then deposits approximately 300 eggs per day in the venous vessels.
The immunoreaction of the host to the parasite eggs which will lodge in the tissues is the principal responsable for the spread of the disease. In some cases, it develops into the acute clinical form with intense toxemic manifestations and, in the majority of victims, a debilitating chronic disease, with possibilities of serious and frequently fatal complications.
Amongst the measures to control the infection, the treatment of the infected, the control of the molluscs, the improvement of living conditions and the identification followed by the early treatment of populations at risk have proven to be the most sustainable, but are by no means completely satisfactory. After the seventies, with the advent of drugs usable on a major scale, the specific therapeutic began to play a crucial role in all programs to control the disease.
The method of diagnosis constitutes a primordial instrument for the programs to control the endemic. It is highly desirable to develop techniques that are more efficient than those presently available.
The diagnosis of the infection can be done through confirmation by microscopy of the presence of parasite eggs in the faeces or urine, depending on the species. The Kato-Katz method (KATZ, N., A. Chaves, J. Pellegrino, 1972, A simple device for quantitative stool thick smear technique in Schistosomiasis mansoni. Rev. Inst. Med. Trop. Sxc3xa3o Paulo. 14: 337-340) is the technique that offers the best conditions of efficiency, cost and operation; (WHO, 1994, The Control of schistosomiasis. Technical Report Series, 830, Geneva, 86 pp.). However, when the quantity of eggs in the faeces is small, such as in conditions of low prevalence and low infection intensity and also when is necessary to monitor the treatment, the sensitivity of the test on a single faecal sample is diminished making a greater number of faecal samples examinations necessary. Even so, some victims may not be diagnosed, and consequently, not receive treatment.
The serological diagnosis constitutes an alternative to the parasitological test, being, after the latter, the second most widely employed. By this method, antibodies produced by infection with S. mansoni are detected by techniques such as the reaction of indirect immunofluorescence and ELISA (Enzyme Linked ImmunoSorbent Assay). However, this technique has the disadvantage of not distinguishing between active infection and past infection. Thus, the low specificity of antibodies against S. mansoni in the absence of active infection is explained by crossed reactivity with other parasites (Corrxc3xaaa-Oliveira R, Dusse L M S, Viana I R C, Colley D G, Carvalho O S, Gazzinelli G 1988. Human antibody response against schistosomal antigens. Am J Trop Med Hyg 38: 348-355) by the presence of unisexual infections, by contact with other cercariae, by the transfer of maternal antibodies and by the persistance of antibodies after a successful previous treatment.
Another alternative for the diagnosis of Schistosoma mansoni consists in detecting antigenic substances released by the parasite, allowing to differentiate between past and active infection and eliminating the problem of the unspecificity of the antibody diagnostic. However, the search for circulating antigens presents other disadvantages, such as low sensitivity to light infections, the high cost, difficulty of operation and a dependency on the production of monoclonal antibodies (D E Jonge,N., A. L. T. Rabello, F. W. Krijger, P. G. Kremsner, R. S. Rocha, N. Katz e A. M. Deelder.1991. Levels of Schistosome circulating anodic and cathodic antigens in serum of Schistosomiasis patients from Brazil. Trans. R. Soc. Trop. Med. Hyg.85:756-759).
With the emergence of Polymerase Chain Reaction (PCR), it has become possible to amplify the DNA of pathogenic organisms enabling that, from minute initial quantities, sufficiently large quantities be obtained to the point of permitting their detection. Thus, PCR has come to be used as an efficient method of diagnosis for the most diverse infections.
Document EP 550 883 describes an experiment using PCR to detect, in faecal matter, the presence of the DNA of protozoan G. lamblia. The procedure was carried out using, as target, the sequence of the gene RNA 18S of G. lamblia. Furthermore the applicant designed oligonucleotide primers sufficiently complementary to a region of the target sequence of the ribosomic RNA 18S of G. lamblia. 
Document WO 94/04681 is related to the development of specific oligonucleotides to be used as primers of the PCR for the detection of the DNA sequence encoding the protein of the wall of Cryptosporidium sp., and thus as a kit for the diagnostic of parasites of this genera. Apart from the above mentioned examples, it is possible to cite the document WO 93/03167, which is an application of a process for the isolation, storage and cleavage of DNA capable of furnishing it in an adequate form for amplification through a specific sequencial process, such as PCR. The process is particularly employed for the detection of parasitic diseases caused by micro-organisms having concatenated closed circular kinetoplast DNA such as T. cruzi, Leishmania sp., P. falciparum, P. vivax and P. malariae. 
In the study of the Schistosoma sp. PCR has been used for determining the sex of the cercariae (Gasser, R. B., G. Morahan e G. F. Mitchell. 1991. Sexing single larval stages of Schistosoma mansoni by polimerase chain reaction. Mol. Bioch. Parasitol. 47:255-258.) in the cloning and sequencing of specific genes (Francis, P. e Q. Bickl.1992. Cloning of a 21.7 kDa vaccine-dominant antigen gene of Schistosoma mansoni reveals an Elf Hand-like motif. Mol. Bioch. Parasitol. 50:215-224.; Kiang, D., A. M. Karim, P. T. LoVerde.1996. Cloning the gene encoding Schistosoma mansoni p50, an immunophilin. Gene. 170:137-140.), in order to ascertain the populational genetic variability of strains of Schistosoma sp. (Dias Neto, E., C. P. Souza, D. Rollinson, N. Katz, and A. J. G. Simpson. 1993. The random amplification of polymorphic DNA allows the identification of strains and species of Schistosomes. Molecular and Biochemical Parasitology 57 (1): 83-88.; Simpson,A. J., E. Dias Neto, T. H. Vidigal, H. B. Pena, O. S. Carvalho e S. D. Pena.1995. DNA polymorphism of schistosomes and their snail hosts. Mem. Inst. Oswaldo Cruz.90(2):211-213.) and in the development and application of techniques for the generation of Expressed Sequence Tags (or EST""s) (Dias Neto, E.; Harrop, R., Correia-Oliveira, R., Pena,S. D. J., Wilson,R. A. e Simpson,A. J. G.1996. The schistosome genome project: RNA arbitrarily primed PCR allows the accelerated generation of expressed sequence tags. Mem. Ist. Oswaldo Cruz. 91(5):655-657; Dias Neto, E., Harrop, R., Correa-Oliveira, R., Wilson, R. A., Pena, S. D. J. e Simpson, A. J. G. 1997. Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the efficient generation of ESTs from nanogram quantities of mRNA. Gene 186: 135-142). Recently, Hamburger et al. (Hamburger, J., X. Yu-Xin, R. M. Ramzy, J. Jourdane e A. Ruppel. 1998. Development and evaluation of a Polymerase Chain Reaction for monitoring Schistosoma mansoni infestation of water. Am. J. Trop. Med. Hyg. 59(3):468-473) developed a PCR-based test to monitor S. mansoni infestation of natural water.
The primers described in the present invention were designed based on the S. mansoni sequence originally described by Hamburger et al. shown in FIG. 1, (Hamburguer, J, Turetski, T, Kapeller, I and Deresiewicz, R. 1991. Highly repeated short DNA sequences in the genome of Schistosoma mansoni recognized by a species-specific probe. Molecular and Biochemical Parasitology 44: 73-80). The design of the primers was done after the visual inspection of the original sequence, so as to avoid the presence of complementary regions within each initiator or between both primers. In order to facilitate the amplification of the degraded DNA molecules (as expected for free DNA present in faeces or body fluids) the primers were chosen in a manner so as to amplify a short sequence. Also, all the stages of the present invention were strictly standardised in a manner as to function satisfactorily with chemically complex biological samples such as faeces and serum.
To the present moment, the use of PCR as a method for diagnosing human Schistosomiasis has never been related.
As such, it becomes evident that the search for a rapid and efficient method for the detection of the Schistosoma sp. parasite, specially useful in cases of low infection intensity, has not yet been viable. Thus, aiming to attain this objective, a strategy for PCR employing primers complementary to a specific and highly repetitive sequence of the S. mansoni genome, is used.
The object of the present invention is the detection of Schistosoma sp. in biological samples by PCR.
A first embodiment of the present invention refers to a method for the diagnosis of Schistosoma through the amplification by PCR of a DNA sequence of Schistosoma sp. The method of the present invention is characterised by the stages of:
(a) collection of the sample to be tested;
(b) extraction of the Schistosoma sp. DNA from the sample obtained in stage (a);
(c) amplify a region of the Schistosoma DNA extracted in stage (b) with specific primers constructed from the original sequence described in SEQ ID NO:1;
(d) separate the products of the amplifications of stage (c) by electrophoresis, followed by detection using appropriate colouring methods.
In a second embodiment, the invention is directed to a diagnostic kit to be used in the detection of Schistosoma. A basic kit includes all the reagents necessary for carrying out the PCR technique, namely: specific primers, nucleotides and appropriate buffer solution for the amplification by PCR. Optionally, the kit may contain the enzyme Taq polymerase in quantities sufficient for amplification, standard DNA to be used as positive control of the reaction, buffer solution of the sample to prepare the amplified material for electrophoresis and protocol and instructions manual for the user.